Biomarkers for cancer play key roles in both the clinic and the lab, providing diagnostic and prognostic tools to enable effective medical interventions, as well as improving our basic understanding of diseases. Clinical applications include early disease detection, prediction of risk, determination of treatment options, and monitoring disease progression and treatment efficacy. However, reaching the point of clinical utility requires overcoming two large hurdles 1) discovering and validating biomarkers, and 2) developing clinical tests to monitor the biomarkers. Limitations of current biomarker detection assays ? including lack of sensitivity, specificity, and clinical compatibility ? are critical obstacles in realizing the promise of cancer biomarkers. We will develop a new approach that will overcome these challenges by leveraging recent developments in DNA nanotechnology and multiplexed single-molecule analysis to create an ultrasensitive detection platform that is low cost, accessible, and capable of detecting a wide variety of both protein and RNA biomarkers in biological fluids. Our approach pairs rationally designed DNA nanoswitches with an accessible new single- molecule instrument to enable high-sensitivity, multiplexed biomarker detection in a benchtop centrifuge. Our recently invented DNA nanoswitches are designed to undergo a conformational change upon binding a specific molecular biomarker, and can be easily ?reprogrammed? for a variety of biomarkers. The induced conformational changes are detected at the single-molecule level using a newly developed Centrifuge Force Microscope (CFM), which is capable of monitoring thousands of such changes simultaneously within a standard benchtop centrifuge. To demonstrate our approach, we will use pancreatic cancer as a model system, where recent evidence has shown advantages in using both protein and microRNA biomarkers for disease diagnosis. Specifically, we will: 1) Demonstrate proof of concept detection and quantification of pancreatic cancer biomarkers CA19-9 and miR-196a, 2) Optimize the biosensing platform for use with complex biological fluids, such as serum and urine, and 3) Multiplex our system to enable the simultaneous detection of multiple RNA and protein biomarkers for pancreatic cancer. Our proposed approach offers a number of important advantages including: i) unambiguous ?digital? on and off signals, ii) single-molecule sensitivity, iii) programmability of detection elements for different biomarkers, iv) simultaneous detection of multiple biomarkers in the same sample, and iv) high accessibility owing to the use of standard laboratory equipment. This biosensing platform has the potential to be a transformative technology in biomedical research and medicine. Single-molecule detection provides the ultimate sensitivity, and our new CFM design enables such sensitivity using already standard lab equipment. By providing detection sensitivity that meets or exceeds state-of-the-art clinical analyzers, we anticipate that our approach could help facilitate new initiatives in precision medicine, such as companion diagnostics for individualized cancer treatments.